Mutant carbamoylphosphate synthetase and method for producing compounds derived from carbamoylphosphate

ABSTRACT

L-arginine, citrulline and pyrimidine derivatives including orotic acid, uridine, uridine 5′-monophosphate (UMP), cytidine and cytidine 5′-monophosphate (CMP) are produced using a bacterium belonging to the genus  Escherichia  harboring a mutant carbamoylphosphate synthetase in which the amino acid sequence corresponding to positions from 947 to 951 in a wild type carbamoylphosphate synthetase is replaced with any one of amino acid sequences of SEQ ID NOS: 1 to 9, and feedback inhibition by uridine 5′-monophosphate in the bacterium is desensitized.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to microbiological industry, specificallyto a method for producing compounds derived from carbamoylphosphate.More specifically, the present invention concerns the using of newfeedback-resistant enzymes involved in arginine and pyrimidinebiosynthesis pathways of E.coli strains producing compounds derived fromcarbamoylphosphate, such as arginine, citrulline and pyrimidinederivatives including orotic acid, uridine, uridine 5′-monophosphate(UMP), cytidine and cytidine 5′-monophosphate (CMP).

2. Description of the Related Art

The carbamoylphosphate synthetase (CPSase) of E.coli catalyzes thecomplex synthesis of carbamoylphosphate (CP) from bicarbonate, glutamineand two molecules of Mg-ATP, with the release of glutamate, phosphate,and two Mg-ADP [Meister A., Advan. Enzymol. Mol. Biol., vol. 62, p.315–374, 1989]. The synthesis of CP is intermediate for two biosyntheticpathways, namely those of pyrimidine nucleotides and arginine. In thefirst pathway, CP is coupled to aspartate carbamoyltransferase (ATCase),resulting in formation of orotate in two steps. Orotate is an importantmetabolic intermediate for the biosynthesis of pyrimidine derivatives,including pyrimidines, such as uracil; pyrimidine nucleosides, such asorotidine, uridine, and cytidine; and pyrimidine nucleotides, such asorotidine 5′-monophosphate (OMP), UMP, and CMP. It was shown that thepresence of orotate in a culturing medium during fermentation of thewide scope of bacteria assists measurably in the production andaccumulation of pyrimidine derivative, namely, uracil (U.S. Pat. No.3,214,344). In the second pathway, CP is coupled to ornitine viaornitine carbamoyltransferase (OTCase), constituting the sixth step(starting from glutamate) in the arginine biosynthetic pathway. CPSaseis activated by ornitine and IMP (a precursor of purine nucleotides) andinhibited by UMP. Carbamoylphosphate synthetase consists of twosubunits. It has been known for coryneform bacteria (EP1026247A1) andfor bacteria belonging to the genera Escherichia and Bacillus that thosesubunits are encoded by carA and carB genes. Transcription of the carABoperon is cumulatively repressed by the end-products of both pathways[Charlier D., et al., J. Mol. Biol., vol. 226, p. 367–386, 1992; WangH., et al., J. Mol. Biol., vol. 277, p. 805–824, 1998; Glansdorff N., etal., Paths to Pyrimidines, vol. 6, p. 53–62, 1998]. The native E.coliCPSase is a heterodimer composed of a small subunit of 41,270 Da and alarge subunit of 117,710 Da, encoded by carA and carB genesrespectively. The small subunit catalyzes the hydrolysis of glutamineand is responsible for the transfer of NH₃ to the large subunit, wherethe CP synthesis actually takes place. The large subunit contains thebinding sites for the substrates bicarbonate, ammonia, two separatesites for Mg-ATP and a 18 kDa carboxyterminal region which constitutesthe regulatory domain [Rubio V., et al., Biochemistry, vol. 30, p.1068–1075, 1991; Cervera J., et al., Biochemistry, vol. 35, p.7247–7255, 1996]. Further, it is suggested that the large subunit has anactivity to catalyze solely a synthetic reaction of carbamoylphosphate(Stephen D. Rubino et al., J. Biol. Chem., 206, 4382–4386, 1987).

The crystal structure of an allosterically activated form of CPSase hasrecently been described [Thoden J., et al., Biochemistry, vol. 36, p.6305–6316, 1997; Thoden J., et al., Acta Crystallogr. Sec. D., vol. 55,p. 8–24, 1999]. The first three distinct domains in the large subunitlabeled as A, B, C are very similar in terms of structure, but thefourth one is entirely different. The D domain (residues 937–1073) isresponsible for the binding and allosteric regulation by effectors: IMP,UMP and ornitine. Also it was shown, that two residues, serine 948 andthreonine 1042, appear to be crucial for allosteric regulation of CPSase[Delannay S., et al., J. Mol. Biol., vol. 286, p. 1217–1228, 1999]. Whenserine 948 is replaced with phenylalanine, the enzyme becomesinsensitive to UMP and IMP, but still activated by ornitine, although toa reduced extent. The enzyme with T1042I mutation displays a greatlyreduced activation by ornitine.

As a rule, the feed back resistance (fbr) phenotype of enzyme arises asa result of the replacing the amino acid residue with another in asingle or in a few sites of amino acid sequence and these replacementslead to reducing the activity of enzyme. For example, the replacing ofnatural Met-256 with each of 19 other amino acid residues in E.coliserine acetyltransferase (SAT) (cysE gene) leads in most cases to fbrphenotype but the mutant SAT proteins do not restore the level ofactivity of natural SAT [Nakamori S. et al. AEM, vol. 64, p. 1607–1611,1998]. So, the disadvantage of the mutant enzymes obtained by thesemethods is the reduced activity of mutant enzymes in comparison with thewild type enzymes.

SUMMARY OF THE INVENTION

The present invention is concerning the construction of feedbackresistant and high active enzymes playing a key role in biosynthesis ofpyrimidines and arginine or citrulline in E.coli.

In the present invention the novel procedure for synthesizing a largeset of mutant carB genes using the full randomization of carB genefragment is proposed. The simultaneous substitutions of some amino acidresidues in fragment of amino acid sequence, in which the fbr mutationcan be localized, can produce mutant proteins with restored level ofactivity close to the natural due to more correct accordance of threedimension structure of enzyme. Thus the present invention describedbelow has been accomplished. That is the present invention provides:

-   (1) A large subunit of the carbamoylphosphate synthetase wherein the    amino acid sequence corresponding to the positions from 947 to 951    of SEQ ID NO: 20 is replaced with any one of amino acid sequences of    SEQ ID NOS: 1 to 9, and feedback inhibition by uridine    5′-monophosphate is desensitized;-   (2) The large subunit of the carbamoylphosphate synthetase according    to (1), wherein the carbamoylphosphate synthetase is that of    Escherichia coli.-   (3) The large subunit of the carbamoylphosphate synthetase according    to (1), wherein the amino acid sequence of the positions from 947 to    951 of SEQ ID NO: 20 is replaced with any one of amino acid    sequences of SEQ ID NOS: 1 to 9, and feedback inhibition by uridine    5′-monophosphate is desensitized;-   (4) The large subunit of the carbamoylphosphate synthetase according    to (1), which includes deletion, substitution, insertion, or    addition of one or several amino acids at one or a plurality of    positions other than the positions from 947 to 951, wherein feedback    inhibition by uridine 5′-monophosphate is desensitized;-   (5) A carbamoylphosphate synthetase which comprises the large    subunit of the carbamoylphosphate synthetase according to any one    of (1) to (4);-   (6) A DNA coding for the carbamoylphosphate synthetase according to    any one of (1) to (4), wherein feedback inhibition by uridine    5′-monophosphate is desensitized;-   (7) A DNA coding the large subunit of carbamoylphosphate synthetase    wherein feedback inhibition by uridine 5′-monophosphate is    desensitized according to any one of (1) to (4), and a small subunit    of carbamoylphosphate synthetase of Escherichia coli.;-   (8) A bacterium belonging to the genus Escherichia, which harbors    the DNA according to (6) or (7);-   (9) The bacterium according to (8), which has an ability to produce    a compound selected from the group consisting of L-arginine,    citrulline and pyrimidine derivatives;-   (10) The bacterium according to (9), wherein the pyrimidine    derivatives are orotic acid, uridine, UMP, cytidine and CMP;-   (11) A method for producing the compound which is selected from the    group consisting of L-arginine, citrulline and pyrimidine    derivatives, which method comprises the steps of cultivating the    bacterium according to any of (8) to (10) in a medium to produce and    accumulate the compound in the medium and collecting compound from    the medium; and-   (12) The method as defined in (11), wherein the pyrimidine    derivatives are orotic acid, uridine, UMP, cytidine and CMP.

In the present invention, the term “CPSase activity” means activity tocatalyze the reaction of the complex synthesis of carbamoylphosphatefrom bicarbonate, glutamine and two molecules of Mg-ATP. The “CPSase” ofthe present invention may be a single polypeptide consisting of thelarge subunit, or may be a heterodimer comprising the large subunit andthe small subunit, provided that the CPSase has the CPSase activity. Inthe present application, the large subunit and the heterodimer asmentioned above may be generically referred to as “CPSase”. A DNAencoding the large subunit and the small subunit may be referred to as“carAB”.

The CPSase having any of fbr mutation as described above may be referredto as “the mutant CPSase”, a DNA coding for the mutant CPSase may bereferred to as “the mutant carB genes” or “the mutant carAB genes”according to the embodiment, and a CPSase without the mutation may bereferred to as “a wild type CPSase”.

Hereafter, the present invention will be explained in detail.

<1> Mutant CPSase and Mutant carB Gene

Subsequent selection and screening of recombinant clones carrying mutantcarB genes cloned as carAB operon into expression vector allows tochoose the fbr variants of mutant CPSase with different level of itsbiological activity.

According to the data obtained by S. Delannay et al. (Delannay S., etal., J. Mol. Biol., v. 286, 1217–1228, 1999) the mutant (S948F) ofcarbamoylphosphate synthase of E.coli is insensitive to UMP. Based onthese data, the region including position 948 in CPSase was selected forthe target of modification.

The mutant CPSase and the mutant carB gene are obtained by randomizedfragment-directed mutagenesis. To obtain the numerous mutations in carBgene, the randomization of 15-nucleotide fragment of carB gene whichcodes for the region from Leu947 to Glu951 residues in the amino acidsequence SEQ ID NO: 20 is carried out (see below). The randomized15-nucleotide fragment gives 4¹² or near 1.5×10⁷ different DNA sequenceswhich can code for 4×10⁵ different amino acid residues in the 5-merpeptide. The likelihood of in frame non-introducing the stop codons inthese sequences is about 0.95 or 95%. So, the randomization of the carBgene fragment coding for the peptide from 947-th to 951-th amino acidresidues must give approximately 4×10⁵ different amino acid sequenceswith diversity in this peptide fragment of CPSase structure. Subsequentselection and screening of recombinant clones carrying mutant carB genescloned into expression vector allows to choose the fbr variants ofmutant CPSases with different level of its biological activity.

The amino acid sequences of the mutant CPSase suitable for fbr phenotypeof CPSase are defined by the present invention. Therefore, the mutantCPSase can be obtained based on the sequences by introducing mutationsinto a wild type carB gene using ordinary methods. As a wild type carBgene, the carB gene of E. coli can be mentioned (nucleotide numbers10158 to 13379 in the sequence of GenBank Accession AE000113 U00096: SEQID NO: 19). The carA gene corresponds to nucleotide numbers 8992 to10140 in the sequence of GenBank Accession U00096.

In the case that the carB gene is used for a material to obtain a DNAencoding the mutant CPSase, the mutant carB gene encoding the largesubunit of the mutant CPSase. In the case that the carAB genes are usedfor the material, the mutant carAB gene encoding the large subunit ofthe mutant CPSase together with the small subunit.

The amino acid sequence of positions from 947 to 951 in the mutantCPSase of the present invention is any one of the sequence of SEQ IDNOS: 1 to 9. The corresponding amino acid sequence of known fbr CPSase,in which Ser at a position 948 is replaced with Phe, and the wild typeCPSase of E. coli are illustrated in Table 1. Examples of nucleotidesequence encoding these amino acid sequences are also shown in Table 1.

TABLE 1 Sequence of DNA sequence of randomized region of SEQ randomizedSEQ No of CarB protein ID fragment of carB ID clone (947→951 a.a.) NO:gene (5′→3′) NO: Wt -Leu-Ser-Val-Arg-Glu- 28 CTTTCCGTGCGCGAA 30 6-Leu-Phe-Val-Arg-Glu- 29 CTTTTCGTGCGCGAA 31 (sin- gle muta- tion) 10-Pro-Leu-Arg-Glu-Gly- 1 CCTCTCCGTGAGGGT 10 12 -Ala-Val-Ala-Leu-Lys- 2GCTGTCGCTTTGAAA 11 13 -Gly-Val-Phe-Leu-Met- 3 GGTGTCTTCCTAATG 12 27-Phe-Phe-Cys-Phe-Gly- 4 TTTTTCTGTTTTGGG 13 31 -Pro-Thr-Gly-Arg-Arg- 5CCTACCGGTAGGAGA 14 33 -Phe-Ala-Cys-Gly-Val- 6 TTCGCCTGTGGGGTG 15 34-Val-Phe-Gly-Ser-Ser- 7 GTTTTCGGTAGTAGT 16 36 -Ala-Ser-Gly-Val-Glu- 8GCTTCCGGCGTTGAG 17 37 -Ala-Phe-Cys-Gly-Val- 9 GCCTTCTGTGGGGTG 18

The mutant CPSase may include deletion, substitution, insertion, oraddition of one or several amino acids at one or a plurality ofpositions other than 947th to 951st. provided that the CPSase activityis not deteriorated. The number of “several” amino acids differsdepending on the position or the type of amino acid residues in thethree dimensional structure of the protein. This is because of thefollowing reason. That is, some amino acids have high homology to oneanother and the difference in such an amino acid does not greatly affectthe three dimensional structure of the protein. Therefore, the mutantCPSase of the present invention may be one which has homology of notless than 30 to 50%, preferably 50 to 70% with respect to the entireamino acid residues for constituting CPSase, and which has the fbrCPSase activity. The mutant CPSase desirably maintain the CPSaseactivity of not less than 25%, preferably not less than 30%, morepreferably not less than 40% of the activity of the wild type CPSase inthe presence of uridine 5′-monophosphate.

In the present invention, “amino acid sequence corresponding to thesequence of positions from 947 to 951” means an amino acid sequencecorresponding to the amino acid sequence of positions from 947 to 951 inthe amino acid sequence of SEQ ID NO: 20. A position of amino acidresidue may change. For example, if an amino acid residue is inserted atN-terminus portion, the amino acid residue inherently locates at theposition 947 becomes position 948. In such a case, the amino acidresidue corresponding to the original position 947 is designated as theamino acid residue at the position 947 in the present invention.

The phrase “feedback inhibition by uridine 5′-monophosphate isdesensitized” means that the degree of the feedback inhibition islowered. The lowering of the degree of feedback inhibition can bedetermined by measuring the lowering of the CPSase activity in thepresence of uridine 5′-monophosphate and by comparing it with that ofprotein having the amino acid sequence of SEQ ID NO: 20. Further, thephrase “feedback inhibition by uridine 5′-monophosphate is desensitized”means that substantial desensitization of inhibition is sufficient, andcomplete desensitization is not necessary. Concretely, it is desirablethat the ratio of the activity of the mutant CPSase in the presence of10 mM uridine 5′-monophosphate to the activity in the absence of uridine5′-monophosphate is not less than 50%, preferably not less than 70%,more preferably not less than 90%, when 5 mM glutamine is used for asubstrate.

The DNA, which codes for the substantially same protein as the mutantCPSase described above, may be obtained, for example, by modifying thenucleotide sequence, for example, by means of the site-directedmutagenesis method so that one or more amino acid residues at aspecified site involve deletion, substitution, insertion, or addition.DNA modified as described above may be obtained by the conventionallyknown mutation treatment. The mutation treatment includes a method fortreating a DNA containing the mutant carB gene in vitro, for example,with hydroxylamine, and a method for treating a microorganism, forexample, a bacterium, belonging to the genus Escherichia harboring themutant carB gene with ultraviolet irradiation or a mutating agent suchas N-methyl-N′-nitro-N-nitrosoquanidine (NTG) and nitrous acid usuallyused for the such treatment.

The substitution, deletion, insertion, or addition of nucleotide asdescribed above also includes mutation, which naturally occurs (mutantor variant), for example, on the basis of the individual difference orthe difference in species or genus of bacterium, which harbors CPSase.

The DNA, which codes for substantially the same protein as the mutantCPSase, can be obtained by isolating a DNA which hybridizes with DNAhaving known carB gene sequence or part of it as a probe under stringentconditions, and which codes for a protein having the CPSase activity,from a cell harboring the mutant CPSase which is subjected to mutationtreatment.

The term “stringent conditions” referred to herein means a conditionunder which so-called specific hybrid is formed, and non-specific hybridis not formed. It is difficult to express this condition precisely byusing any numerical value. However, for example, the stringentconditions include a condition under which DNAs having high homology,for example, DNAs having homology of not less than 50% with each otherare hybridized, and DNAs having homology lower than the above with eachother are not hybridized. Alternatively, the stringent condition isexemplified by a condition under which DNA's are hybridized with eachother at a salt concentration corresponding to an ordinary condition ofwashing in Southern hybridization, i.g., 60° C., 1×SSC, 0.1% SDS,preferably 0.1×SSC, 0.1% SDS.

The gene, which is hybridizable under the condition as described above,includes those having a stop codon generated within a coding region ofthe gene, and those having no activity due to mutation of active center.However, such inconveniences can be easily removed by ligating the genewith a commercially available expression vector, and investigatingCPSase activity of expressed protein.

When CPSase of the present invention is a heterodimer comprising themutant large subunit and the small subunit, the small subunit isexemplified by a small subunit of a wild type CPSase of Escherichiacoli.

In the present invention, the small subunit may include deletion,substitution, insertion, or addition of one or several amino acids atone or a plurality of positions, provided that it shows the CPSaseactivity together with the large subunit. The meaning of the term“several” is the same as described above for the large subunit.

As the DNA encoding the substantially same polypeptide as the smallsubunit described above, a DNA which is hybridizable to DNA containingcarA or a part thereof under the stringent conditions can be mentioned.The meaning of the term “stringent conditions” is the same as describedabove.

<2> Bacterium Belonging to the Genus Escherichia of the PresentInvention.

The bacterium belonging to the genus Escherichia of the presentinvention is a bacterium belonging to the genus Escherichia to which themutant carB gene described above is introduced. A bacterium belonging tothe genus Escherichia is exemplified by E. coli. The mutant carB genecan be introduced by, for example, transformation of a bacteriumbelonging to the genus Escherichia with a recombinant DNA comprising avector which functions in a bacterium belonging to the genus Escherichiaand the mutant carB gene. The mutant carB gene can be also introduced bysubstitution of carB gene on a chromosome with the mutant carB gene.

Vector using for introduction of the mutant carB gene is exemplified byplasmid vectors such as pBR322, pMW118, pUC19 or the like, phage vectorssuch as 11059, 1BF101, M13mp9 or the like and transposon such as Mu,Tn10, Tn5 or the like.

The introduction of a DNA into a bacterium belonging to the genusEscherichia can be performed, for example, by a method of D. A. Morrison(Methods in Enzymology, 68, 326 (1979)) or a method in which recipientbacterial cell are treated with calcium chloride to increasepermeability of DNA (Mandel, M., and Higa, A., J. Mol. Biol., 53, 159,(1970)) and the like.

A produced amount of compound derived from carbamoylphosphate, such asL-arginine, citrulline and pyrimidine derivatives, can be increased byintroduction of the mutant carB gene into producing bacterium belongingto the genus Escherichia as described above. Besides, an ability toproduce compounds, such as L-arginine, citrulline and pyrimidinederivatives, may be imparted to a bacterium to which the mutant carBgene is introduced previously. The pyrimidine derivatives above includeorotic acid, uridine, UMP, cytidine and CMP.

As the bacteria belonging to the genus Escherichia which have anactivity to produce L-arginine are exemplified by E. coli strainsAJ11531 and AF11538 (JP56106598A2), AJ11593 (FERM P-5616) and AJ11594(FERM P-5617) (Japanese Patent Laid-open No. 57-5693), VKPM B-7925(Russian Patent Application No. 2000117677). The strain VKPM B-7925 hasbeen deposited in the Russian National Collection for IndustrialMicroorganisms (VKPM) since Apr. 10, 2000.

L-citrulline producing bacteria belonging to the genus Escherichia,orotate producing bacteria belonging to the genus Escherichia anduridine 5′-monophosphate (UMP) producing bacteria belonging to the genusEscherichia are not known at present.

As the bacteria belonging to the genus Bacillus which have an activityto produce L-citrulline are exemplified by B. subtilis strains K-X-1 A-1(ATCC No 15561) and K-X-1 A-9 (ATCC No 15562) (U.S. Pat. No. 3,282,794),Bacillus sp. strain cit-70 (Japanese Laid-open patent application No.08-089269). The bacteria belonging to the genus Brevibacterium, whichhave an activity to produce L-citrulline, are exemplified byBrevibacterium flavum strains AJ3408 (FERM P-1645) (U.S. Pat. No.5,164,307) and AJ11677 (Japanese Laid-open patent application No.57-163488). The bacterium belonging to the genus Corynebacterium, whichhas an activity to produce L-citrulline, is exemplified byCorynebacterium glutamicum strain AJ11588 (FERM P-5643) (U.S. Pat. No.5,164,307).

As the bacterium belonging to the genus Bacillus which has an activityto produce orotic acid is exemplified by B. subtilis strain FERMP-11402, deficient in orotate phosphoribosyltransferase (JapaneseLaid-open patent application No. 04-004891). The bacteria belonging tothe genus Corynebacterium which have an activity to produce UMP areexemplified by Corynebacterium glutamicum strains T-26 (FERM BP-1487),resistant to 5-fluorouracyl, T-29 (FERM BP-1488), resistant to5-fluorouracyl and trimethoprim, and T-30 (FERM BP-1489), resistant to5-fluorouracyl and sulfaguanidine (European patent EP0312912).

The bacteria belonging to the genus Corynebacterium which have anactivity to produce UMP are exemplified by Corynebacterium ammoniagenesstrains LK 40-2 (VKPM B-7811), LK 75-15 (VKPM B-7812), and LK 75-66(VKPM B-7813) (Russian patent application No. 99122774).

<3> Method for Producing L-arginine, Citrulline and PyrimidineDerivatives.

Compounds, such as L-arginine, citrulline and pyrimidine derivatives,can be efficiently produced by cultivating the bacterium to which themutant carB gene is introduced and which has an ability to produce saidcompounds in a culture medium, producing and accumulating said compoundsin the medium, and collecting them from the medium. The pyrimidinederivatives above include orotic acid, uridine, UMP, cytidine and CMP.In the method of present invention, the cultivation of the bacteriumbelonging to the genus Escherichia, the collection and purification ofcompounds from the liquid medium may be performed in a manner similar tothose of the conventional method for producing L-arginine byfermentation using a bacterium. A medium used in cultivation may beeither a synthetic medium or a natural medium, so long as the mediumincludes a carbon and a nitrogen source and minerals and, if necessary,nutrients the bacterium used requires for growth in appropriate amount.The carbon source may include various carbohydrates such as glucose andsucrose, and various organic acids, depending on assimilatory ability ofthe used bacterium. Alcohol including ethanol and glycerol may be used.As the nitrogen source, ammonia, various ammonium salts as ammoniumsulfate, other nitrogen compounds such as amines, a natural nitrogensource such as peptone, soybean hydrolyzate and digested fermentativemicrobe may be used. As minerals, monopotassium phosphate, magnesiumsulfate, sodium chloride, ferrous sulfate, manganese sulfate, calciumcarbonate may be used.

The cultivation is preferably the one under an aerobic condition such asa shaking, and aeration and stirring culture. The cultivation is usuallyperformed at a temperature between 20 and 40° C., preferably 30 and 38°C. The cultivation is usually performed at a pH between 5 and 9,preferably between 6.5 and 7.2. The pH of the culture medium can beadjusted with ammonia, calcium carbonate, various acids, various bases,and buffers. Usually, a 1 to 3-day cultivation leads to the accumulationof the compounds in the medium.

The isolation of the compounds can be performed by removing solids suchas cells from the medium by centrifugation or membrane filtration aftercultivation, and then collecting and purifying such compounds by ionexchange, concentration and crystalline fraction methods and the like.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows scheme of construction the plasmid pEL-carAB-wt.

FIG. 2 shows scheme of construction the pool of mutant carB genes.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be specifically explained with reference tothe following examples.

EXAMPLE 1

The plasmid pBScarAB-13 carrying wild type carAB genes from E.coli wasconstructed by cloning the AvaIII-BglII DNA fragment (4911 bp) fromE.coli chromosome to pBluescript II SK(+) vector (Fermentas, Lithuania)previously digested by BamHI and PstI.

The plasmid pET22-b(+) (Novagen, USA) was modified to substitute the T7promoter by lac promoter. The lac promoter was obtained by PCRamplification using plasmid pUC18 as a template and the oligonucleotides5′-accagatctgcgggcagtgagcgcaacgc-3′ (SEQ ID NO: 21) and5′-gtttctagatcctgtgtgaaattgttatccgc-3′ (SEQ ID NO: 22) as a primers. Theresulted fragment (0.14 kb) carrying the lac promoter was digested byrestrictases BglII and XbaI and cloned into pET22-b(+) vector previouslydigested by the same restrictases. The resulted plasmid pET-Plac wasused for cloning the carAB genes without promoter from plasmidpBScarAB-13.

The 5′-end of carA gene (1.18 kb) was obtained by PCR amplificationusing pBScarAB-13 as a template and oligonucleotides5′-cctctagaaataaagtgagtgaatattc-3′ (SEQ ID NO: 23) and5′-cttagcggttttacggtactgc-3′ (SEQ ID NO: 24) as a primers. The resultedfragment was digested by XbaI and DraIII and XbaI-DraIII fragment (0.61kb) carrying 5′-end sequence of carA gene was purified by agaroseelectrophoresis. The mixture of this fragment, the DraIII-SacI fragmentfrom plasmid pBScarAB-13 (carrying sequences of 3′-end of carA gene andcarB gene) and vector pET-Plac/XbaI-SacI were ligated and transformedinto E.coli TG1 cells. The recombinant plasmid pEL-carAB-wt obtainedcarried sequence of wild type carAB operon under control of lacpromoter.

The TaKaRa La DNA Polymerase used for PCR amplification was obtainedfrom Takara Shuzo Co. (Japan) and was used under the conditionsrecommended by the supplier.

<1> The Randomized Fragment-directed Mutagenesis

The plasmid pBScarAB-13 was used as the template, the sense primer P1:5′-ggtcgtgcgctgNN(T/C)N(T/C)CNN(T/C)NNN(G/A)NNggcgataaagaacgcgtggtg-3′(SEQ ID NO: 25) (48 bases) is designed based on the nucleotide sequenceof carB gene and the standard M13 direct sequence primer is used as aantisense primer. The fixed 12-nucleotide 5′-end and fixed 21-nucleotide3′-end regions of primer P1 are homologous to the sequence of carB genedownstream Glu951 codon and upstream Leu947 codon, respectively.

The 0.75 kbp DNA fragment (3′-end of carB gene) was synthesized duringfirst round of PCR (15 cycles) using the primer P1 with randomized 15nucleotide region. The first round of PCR was performed as follows. 100ng of plasmid pBScarAB-13 were added as a template to PCR solution (50μl) containing each of the two primers at a concentration of 10 pmol.Fifteen PCR cycles (94° C. for 15 sec, 52° C. for 20 sec, 72° C. for 1min) was carried out with a model 2400 DNA thermal cycler (Perkin-ElmerCo., Foster City, Calif.)

At the second round of amplification the next fifteen cycles (94° C. for1 min, 35° C. for 1 min, 72° C. for 2 min) was carried out in which the(−) chain of this fragment is functioning as a “primer” for extension itto get the full gene sequence.

At the third round, the 10 μl aliquot of the reaction mixture was addedto a fresh 90 μl reaction mixture containing 100 pmol of the senseprimer: standard M13 direct sequence primer and primer P2:5′-ccacttcctcgatgacgcgg-3′ (SEQ ID NO: 26) as antisense, and additionalfifteen cycles (94° C. for 0.5 min, 55° C. for 20 sec. 72° C. for 2 min)was performed.

The 1.32 kb DNA fragment coding the pool of mutant variants of 3′-endfragment of carB gene was purified by agarose gel electrophoresis, thenwas digested with AflII and SacI, and further was ligated to thepEL-carAB-wt vector previously digested by the same restrictases toobtain pEL-carAB-NN.

About 150 ng of DNA ligated was used for transformation of E.coli TG1(supE hsdΔ5 thi Δ(lac-proAB) F′[traD36 proAB⁺ lacI^(q) lacZΔM15]) (J.Sambrook et al., Molecular Cloning, 1989) recipient cells in subsequentexperiments to give about 2000 recombinant clones in each case. The poolof recombinant plasmids (pEL-carAB-NN) was purified and transformed intoE.coli cells VKPM B-6969 (carB::Tn10), which were used to select therecombinant plasmids pEL-carAB-NN carrying carAB genes encoding activeCarAB enzymes.

<2> The Site-directed Mutagenesis

For introducing the single mutation Ser948Phe PCR was performed usingthe plasmid pBScarAB-13 as the template, the sense primer5′-cgtgcgctgcttttcgtgcgcgaaggcgataaag-3′ (34 bases) (SEQ ID NO: 27)designed based on the nucleotide sequence of carB gene and the standardM13 direct sequence primer as a antisense primer. The PCR amplificationand cloning of the fragments was carried out as described above.

The 1.32 kbp DNA fragment coding the 3′-end fragment of carB gene withsingle mutation was purified by agarose gel electrophoresis, wasdigested with AflII and SacI. and then was ligated to the pEL-carAB-wtvector previously digested with the same restrictases.

About 100 ng of resulted DNA plasmid was used for transformation ofE.coli cells VKPM B-6969 and the recombinant plasmid pEL-carAB-6carrying active CarAB enzymes with single substitution Ser948Phe wasselected.

EXAMPLE 2 Isolation of New carB Mutants and Effect of Amino AcidSubstitutions in CPSase on Catalytic Properties

At first, the CarAB activity and its feed back resistance to UMP wereevaluated in reactions of biosynthesis of the citrulline from ornithinecatalyzed by CarAB and ArgI (ornitine carbamoyltransferase) enzymes inforty recombinant B-6969(pEL-carAB-NN) clones.

The scheme of reaction is following:

In this reaction carbamoylphosphate synthetase uses free NH₄ ⁺ assubstrate.

The protein extracts from forty B-6969(pEL-carAB-NN) strains andTG1(pUC18-argI) cells were prepared from crude cellular extracts ofsonicated cells by precipitation with (NH₄)₂SO₄ (75% of saturation). Theprotein precipitates were solubilized in buffer of followingcomposition: Tris-HCl (50 mM), pH 7.5, 2-mercaptoethanol (2 mM).

The test-system included the protein extracts from strainsB-6969(pEL-carAB-NN) and TG1(pUC18-argI) and following reagents: ATP (8mM), MgSO₄ (8 mM), (NH₄)₂SO₄ (200 mM), Na₂CO₃ (8 mM) and ornitine (1mM), (pH 7.5). Content of ornitine and citrulline in reaction mixtureswas analyzed by TLC using the liquid phase of following composition:isopropanol/ethyl acetate/ammonium hydroxide/H₂O=40/20/13/27 (v/v).

The 9 clones which expressed the active and feed-back resistant to UMPmutant CPSases and one clone which expressed mutant CPSase with singlesubstitution Ser948Phe were used for measuring the mutant enzymesactivity.

The plasmids from said 10 clones were purified and sequences ofrandomized fragments of carB genes were determined using dideoxy chaintermination method (table 1).

Then, the protein extracts from these 9 clones B-6969(pEL-carAB-NN) andone clone B-6969(pEL-carAB-6) were used to evaluate the activity and fbrof mutant CPSases in reaction of carbamoylphosphate (CP) synthesis fromglutamine or ammonia.

The crude cellular extracts from cells were prepared by sonication of 20mg wet cells pellet suspended in 0,5 ml of buffer A (200 mMK₂HPO₄/KH₂PO₄, pH 8.0, 1 mM EDTA, 1 mM PMSF, 1 mM DTT) and treated withsolid ammonium sulfate to achieve 65% of saturation. After incubationfor 10 min at 4° C., the suspensions were centrifuged at 13,000 rpm for10 min and the precipitates were dissolved in 1 ml of buffer B (20 mMK₂HPO₄/KH₂PO₄, pH 8.0, 50 mM KCl, 1 mM PMSF, 1 mM DTT). The aliquots ofobtained protein extracts were used to evaluate the CPSase activity. Theschemes of reaction were following:

The 50 μl of each reaction mixture included:

reaction I—20 mM tris-HCl, pH 8.0, 100 mM KCl, 5 mM Na₂CO₃, 10 mM ATP,10 mM MgCl₂, 5 mM glutamine, 10 μl of protein extract;

reaction II—20 mM tris-HCl, pH 8.0, 100 mM KCl, 5 mM Na₂Co₃, 10 mM ATP,10 mM MgCl₂, 200 mM (NH₄)₂SO₄, 10 μl of protein extract.

Also, the series of reaction I were carried out in the presence of 10 mMUTP to estimate the level of feed back inhibition of CPSases.

After incubation for 10 min at 37° C. reactions were stopped by additionof equal volume of EtOH, cooled at −20° C. for 10 min and centrifuged at13,000 rpm for 1 min at room temperature. Supernatants were cooled at−20° C.

The CP content in reaction mixtures was analyzed by capillary zoneelectrophoresis. The separation was performed on a Quanta 4000ECapillary Electrophoresis System (“Waters”, USA) with UV indirectdetection at 254 nm. The injection was performed by hydrostatic for 25s. The separation was carried out with an uncoated fused-silicacapillary (75u I.D.* 60 cm, effective length 53 cm) and was operated at−25 kV potential. Temperature was maintained at 20° C. The separationbuffer consisted of 50 mM Tris base, 25 mM benzoic acid (for indirectdetection), pH 8.5, 0.25 mM TTAB (tetradecyl-trimethyl-ammonium bromide)(for reversion of electroosmotic flow).

The data of the measured activity and fbr of mutant CPSases in reactionof CP synthesis are shown in the Table 2.

TABLE 2 The activity of mutant CPSases Activity, (CP, nmol/mg × min)Substrate: 5 mM Gln; No of Substrate: Substrate: Allosteric effector: 10clone 5 mM Gln 200 mM (NH₄)₂SO₄ mM UMP Wt 1350 425 170 6 320 220 320 10690 225 625 12 540 95 540 13 350 60 350 27 730 400 670 31 1120 375 81033 510 150 510 34 765 345 765 36 390 90 390 37 475 205 475

So, the mutated CPSases are essentially insensitive to UMP but thesingle mutation significantly reduced the activity of enzyme. Theseresults indicate that peptide fragment from 947 to 951 amino acidresidues is responsible for the feedback inhibition of CPSase by UMP andfor the level of catalytic efficiency of mutant CPSases as well.

The genes coding the wt CarAB and the mutant CarAB-34 were cloned intoplasmid pMW119. For this purpose, the plasmids pEL-carAB-wt andpEL-carAB-34 were digested by restrictases SacI and XbaI (partialdigestion was used because said plasmids carried two XbaI sites) andfragments coding carAB genes were cloned into pMW119 vector previouslytreated by the same restrictases. As a result, the low-copy numberplasmids pMW119carAB-wt and pMW119carAB-34 carrying carAB genes undercontrol of lac promoter were constructed.

EXAMPLE 3 Production of Orotic Acid Using the Strains Carrying MutantcarAB Genes

The strain 311 was derived from E.coli K12 having insertion of Tn 10into the argA gene (VKPM B-3853) as a mutant strain resistant to6-azauracil (1 mg/ml). The strain 311 has been deposited in the RussianNational Collection of Industrial Microorganisms (VKPM) under theaccession number: VKPM B-8085, since Mar. 5, 2001, and then the originaldeposit was converted to the international deposit on Jul. 17, 2002,according to the provisions of Budapest Treaty.

Strain 311 was transformed with plasmids pMW-carABwt and pMW-carAB-34and production of orotic acid by resulted recombinant strains in thepresence of different concentrations of uridine was tested.

The cultivation conditions in test-tube fermentation was as follows:

1/20 diluted overnight culture, 60 g/l glucose, 25 g/l ammonia sulfate,2 g/l KH₂PO₄, 1 g/l MgSO₄, 0.1 mg/l thiamine, 5 g/l yeast extract Difco,25 g/l chalk, per 1 liter of tap water (pH 7.2). Glucose and chalk weresterilized separately. 2 ml of the medium was placed into test-tubes,and the cultivation was carried out at 32° C. for 3 days with shaking.The level of orotic acid production was evaluated by HPLC (table 3).

TABLE 3 The level of orotic acid production in strains 311 (pMW119), 311(pMW-CarAB-wt), 311 (pMW-CarAB-34) Uridine, 100 Uridine, 300 Uridine,1000 mg/l mg/l mg/l Orotic acid Orotic acid Orotic acid A₅₅₀,biosynthesis, A₅₅₀, biosynthesis, A₅₅₀, biosynthesis, Strain o.u. g/lo.u. g/l o.u. g/l 311 (pMW119) 13.1 0.12 13.8 0.11 9.0 0.01 311 (pMW-11.4 0.27 12.2 0.18 9.8 0.03 CarAB-wt) 311 (pMW- 12.6 0.66 12.7 0.4010.3 0.11 CarAB-34)

As is shown in the Table 3, the strain 311(pMW-CarAB-34) carrying mutantcarAB gene produced more orotic acid compared to the parent strain311(pMW119) and strain 311(pMW-CarAB-wt) carrying wild type carAB gene.

EXAMPLE 4 Production of Arginine and/or Citrulline Using the StrainsCarrying Mutant carAB Genes

The arginine producing strains 333 and 374 have been selected from thederivative of the strain E.coli 57 (VKPM B-7386) having insertion oftransposone Tn 5 into the gene ilvA as mutants resistant to 6-azauracil(1 mg/ml). The strains 333 and 374 have been deposited in the RussianNational Collection of Industrial Microorganisms (VKPM) under theaccession numbers VKPM B-8084 and VKPM B-8086, respectively, since Mar.5, 2001, and then the original deposit was converted to theinternational deposit on Jul. 17, 2002, according to the provisions ofBudapest Treaty.

The strains 333 and 374 were transformed with plasmids pMW-carABwt andpMW-carAB-34 and production of Arg and Cit by these recombinant strainswas tested.

The test-tube fermentation was performed the same manner as in Example3.

The levels of Arg and/or Cit production by strains 333(pMW-CarAB-wt) and333(pMW-CarAB-34) in synthetic medium with 100 mg/l uridine are shown inthe Table 4.

TABLE 4 The levels of Arg and/or Cit production in strains333(pMW-CarAB-wt) and 333(pMW-CarAB-34) Level of Level of Level of ArgCit Arg + Cit Absorbance, biosynthesis, biosynthesis, biosynthesis,Strain A₅₆₀, u. g/l g/l g/l 333(pMW- 24.1 0.60 0.39 0.99 CarABwt)333(pMW- 20.5 1.01 0.51 1.52 CarAB-34)

The level of Cit production by strains 374(pMW-CarAB-wt) and374(pMW-CarAB-34) in synthetic medium with 100 mg/l uridine are shown inthe Table 5.

TABLE 5 The level of Cit production in strains 374(pMW-CarAB-wt) and374(pMW-CarAB-34) Absorbance, Level of Cit Strain A₅₆₀, u. biosynthesis,g/l 374(pMW-CarABwt) 22.3 <0.01 374(pMW-CarAB-34) 15.5 0.26

As is shown in the table 4, the strain 333(pMW-CarAB-34) carrying mutantcarAB gene produced more Arg and Cit than the strains carrying wild typecarAB gene. As is shown in the Table 5, the strain 374(pMW-CarAB-34)produced more Cit than the strains carrying wild type carAB gene.

1. A DNA coding for a mutant of a large subunit of a carbamoylphosphatesynthetase, wherein said mutant has an amino acid sequence of a largesubunit of carbamoylphosphate synthetase as defined in the following (A)or (B), whereby amino acid residues corresponding to positions 947 to951 of SEQ ID NO:20 are replaced with any one of amino acid sequences ofSEQ ID NOs:1 to 9, and is desensitized to feedback inhibition by uridine5′-monophosphate: (A) a protein having the amino acid sequence definedin SEQ ID NO:20; or (B) a protein that is encoded by a DNA whichhybridizes with a DNA having the nucleotide sequence defined in SEQ IDNO:19 under stringent conditions, and has a carbamoylphosphatesynthetase activity, wherein said stringent conditions entail atemperature of 60° C., a salt concentration of 0.1×SSC, and 0.1% SDS. 2.A DNA coding for a mutant of a large subunit of carbamoylphosphatesynthetase, and a small subunit of carbamoylphosphate synthetase ofEscherichia coli, wherein said mutant has an amino acid sequence of alarge subunit of carbamoylphosphate synthetase as defined in thefollowing (A) or (B), whereby amino acid residues corresponding topositions 947 to 951 of SEQ ID NO:20 are replaced with any one of aminoacid sequences of SEQ ID NOs:1 to 9, and is desensitized to feedbackinhibition by uridine 5′-monophosphate: (A) a protein having the aminoacid sequence defined in SEQ ID NO:20; or (B) a protein that is encodedby a DNA which hybridizes with a DNA having the nucleotide sequencedefined in SEQ ID NO:19 under stringent conditions, and has acarbamoylphosphate synthetase activity, wherein said stringentconditions entail a temperature of 60° C., a salt concentration of0.1×SSC, and 0.1% SDS.
 3. A bacterium belonging to the genusEscherichia, which is transformed with the DNA according to claim
 1. 4.The bacterium according to claim 3, which has an ability to produce acompound selected from the group consisting of L-arginine, citrullineand pyrimidine derivatives.
 5. The bacterium according to claim 4,wherein the pyrimidine derivatives are orotic acid, uridine, uridine5′-monophosphate, cytidine and cytidine 5′-monophosphate.
 6. A bacteriumbelonging to the genus Escherichia, which is transformed with the DNAaccording to claim
 2. 7. The bacterium according to claim 6, which hasan ability to produce a compound selected from the group consisting ofL-arginine, citrulline and pyrimidine derivatives.
 8. The bacteriumaccording to claim 7, wherein the pyrimidine derivatives are oroticacid, uridine, uridine 5′-monophosphate, cytidine and cytidine5′-monophosphate.
 9. A method for producing a compound selected from thegroup consisting of L-arginine, citrulline and pyrimidine derivatives,comprising cultivating the bacterium according to claim 3 in a medium toproduce and accumulate the compound in the medium and collecting thecompound from the medium.
 10. The method according to claim 9, whereinthe pyrimidine derivatives are orotic acid, uridine, uridine5′-monophosphate, cytidine and cytidine 5′-monophosphate.
 11. A methodfor producing the compound selected from the group consisting ofL-arginine, citrulline and pyrimidine derivatives, comprisingcultivating the bacterium according to claim 6 in a medium to produceand accumulate the compound in the medium and collecting the compoundfrom the medium.
 12. The method according to claim 11, wherein thepyrimidine derivatives are orotic acid, uridine, uridine5′-monophosphate, cytidine and cytidine 5′-monophosphate.